ABSTRACTIt has been commonly found that in patients presenting Pancreatic Ductal Adenocarcinoma (PDAC), after a period of satisfactory response to standard treatments, the tumor becomes non-responsive and patient death quickly follows. This phenomenon is mainly due to the rapid and uncontrolled development of the residual tumor. The origin and biological characteristics of residual tumor cells in PDAC still remain unclear. In this work, using PDACs from patients, preserved as xenografts in nude mice, we demonstrated that a residual PDAC tumor originated from a small number of CD44+ cells present in the tumor. During PDAC relapse, proliferating CD44+ cells decrease expression of ZEB1, while overexpressing the MUC1 protein, and gain morphological and biological characteristics of differentiation. Also, we report that CD44+ cells, in primary and residual PDAC tumors, are part of a heterogeneous population, which includes variable numbers of CD133+ and EpCAM+ cells. We confirmed the propagation of CD44+ cells in samples from cases of human relapse, following standard PDAC treatment. Finally, using systemic administration of anti-CD44 antibodies in vivo, we demonstrated that CD44 is an efficient therapeutic target for treating tumor relapse, but not primary PDAC tumors. We conclude that CD44+ cells generate the relapsing tumor and, as such, are themselves promising therapeutic targets for treating patients with recurrent PDAC.

Figure 5: Sensitivity of PDX-derived cells in vivo(A) Representative flow cytometry plots for CD44-APC, EpCAM-VioBlue and CD133-FITC in three PDXs treated with vehicle or 100 mg/kg gemcitabine. (B) Quantification of flow cytometry analysis performed in A. (C) Vehicle or Gemcitabine PDX-derived cells were treated with increasing concentrations of Gemcitabine, Docetaxel (TXT), 5-Fluouracil (5FU), Oxaliplatin and the active metabolite of Irinotecan known as SN-38. The rate of cell survival was measured after 72 h of treatment. Error bars ± SEM; n=3 per group. *P<0.05, **P<0.001 compared to vehicle treatment.

Mentions:
Since the expression of CSC markers does not predict the responsiveness to the anticancer treatments, we further studied whether CSC-like cells, present in the residual tumors, are insensitive to the most commonly used PDAC chemotherapeutics. First, we used flow cytometry to measure the percentage of CD44+ cells in primary cultures obtained from X-IPC, C-NOR and AO-IPC PDXs, treated with vehicle or with gemcitabine. We found a significant difference in the proportion of CD44+ cells, from 23.9% to 61.6% for X-IPC, from 22.8% to 65.8% for C-NOR and from 30.4% to 84.4% for AO-IPC, between vehicle and gemcitabine treated PDXs respectively. We also measured expression of EpCAM and CD133 in these primary cultures and found a wide variation between tumors from 0% to 3.5% in vehicle-treated cells and from 0.2% to 22.1% in gemcitabine-treated tumors for EpCAM. The values for CD133 were 1.2% to 3.5% for vehicle-treated cells and from 2.5% to 14.0% for gemcitabine-treated cells (Figure 5A, B). More importantly, we found that almost all of the EpCAM+ and CD133+ cells were included in the CD44+ cell population (Figure S4). Altogether these data allow us to suggest that residual tumors originate mainly from a population of CD44+ cells in PDAC, and that CD44+ cells are a heterogeneous population. We then examined the impact of treatment with increasing doses of gemcitabine, docetaxel, 5FU, oxaliplatin and SN-38 in primary cultured cells, obtained from PDXs, treated with vehicle or with gemcitabine. As expected, treatment with increasing concentration of gemcitabine showed a higher resistance to the drug, with a IC50 ranging from 1 to 15.6 μM, 0.06 to 0.33 μM and 0.015 to 0.06 μM for X-IPC, C-NOR and AO-IPC vehicle or gemcitabine-treated cells, respectively, as showed in Figure 5C. Unexpectedly, chemosensitivity to docetaxel, 5FU, oxaliplatin and SN-38 varied one cell to another for PDX-derived cell population. It should be: Unexpectedly, chemosensitivity to docetaxel, 5FU, oxaliplatin and SN-38 varied from one PDX-derived cell population to another, according to tumor and drug utilized as shown in Figure 5C. These results allow us to conclude that a higher expression of putative CSCs (CD44+) in a residual tumor does not predict the sensitivity to chemotherapeutic treatments in vitro.

Figure 5: Sensitivity of PDX-derived cells in vivo(A) Representative flow cytometry plots for CD44-APC, EpCAM-VioBlue and CD133-FITC in three PDXs treated with vehicle or 100 mg/kg gemcitabine. (B) Quantification of flow cytometry analysis performed in A. (C) Vehicle or Gemcitabine PDX-derived cells were treated with increasing concentrations of Gemcitabine, Docetaxel (TXT), 5-Fluouracil (5FU), Oxaliplatin and the active metabolite of Irinotecan known as SN-38. The rate of cell survival was measured after 72 h of treatment. Error bars ± SEM; n=3 per group. *P<0.05, **P<0.001 compared to vehicle treatment.

Mentions:
Since the expression of CSC markers does not predict the responsiveness to the anticancer treatments, we further studied whether CSC-like cells, present in the residual tumors, are insensitive to the most commonly used PDAC chemotherapeutics. First, we used flow cytometry to measure the percentage of CD44+ cells in primary cultures obtained from X-IPC, C-NOR and AO-IPC PDXs, treated with vehicle or with gemcitabine. We found a significant difference in the proportion of CD44+ cells, from 23.9% to 61.6% for X-IPC, from 22.8% to 65.8% for C-NOR and from 30.4% to 84.4% for AO-IPC, between vehicle and gemcitabine treated PDXs respectively. We also measured expression of EpCAM and CD133 in these primary cultures and found a wide variation between tumors from 0% to 3.5% in vehicle-treated cells and from 0.2% to 22.1% in gemcitabine-treated tumors for EpCAM. The values for CD133 were 1.2% to 3.5% for vehicle-treated cells and from 2.5% to 14.0% for gemcitabine-treated cells (Figure 5A, B). More importantly, we found that almost all of the EpCAM+ and CD133+ cells were included in the CD44+ cell population (Figure S4). Altogether these data allow us to suggest that residual tumors originate mainly from a population of CD44+ cells in PDAC, and that CD44+ cells are a heterogeneous population. We then examined the impact of treatment with increasing doses of gemcitabine, docetaxel, 5FU, oxaliplatin and SN-38 in primary cultured cells, obtained from PDXs, treated with vehicle or with gemcitabine. As expected, treatment with increasing concentration of gemcitabine showed a higher resistance to the drug, with a IC50 ranging from 1 to 15.6 μM, 0.06 to 0.33 μM and 0.015 to 0.06 μM for X-IPC, C-NOR and AO-IPC vehicle or gemcitabine-treated cells, respectively, as showed in Figure 5C. Unexpectedly, chemosensitivity to docetaxel, 5FU, oxaliplatin and SN-38 varied one cell to another for PDX-derived cell population. It should be: Unexpectedly, chemosensitivity to docetaxel, 5FU, oxaliplatin and SN-38 varied from one PDX-derived cell population to another, according to tumor and drug utilized as shown in Figure 5C. These results allow us to conclude that a higher expression of putative CSCs (CD44+) in a residual tumor does not predict the sensitivity to chemotherapeutic treatments in vitro.

Bottom Line:
The origin and biological characteristics of residual tumor cells in PDAC still remain unclear.During PDAC relapse, proliferating CD44+ cells decrease expression of ZEB1, while overexpressing the MUC1 protein, and gain morphological and biological characteristics of differentiation.We confirmed the propagation of CD44+ cells in samples from cases of human relapse, following standard PDAC treatment.

ABSTRACTIt has been commonly found that in patients presenting Pancreatic Ductal Adenocarcinoma (PDAC), after a period of satisfactory response to standard treatments, the tumor becomes non-responsive and patient death quickly follows. This phenomenon is mainly due to the rapid and uncontrolled development of the residual tumor. The origin and biological characteristics of residual tumor cells in PDAC still remain unclear. In this work, using PDACs from patients, preserved as xenografts in nude mice, we demonstrated that a residual PDAC tumor originated from a small number of CD44+ cells present in the tumor. During PDAC relapse, proliferating CD44+ cells decrease expression of ZEB1, while overexpressing the MUC1 protein, and gain morphological and biological characteristics of differentiation. Also, we report that CD44+ cells, in primary and residual PDAC tumors, are part of a heterogeneous population, which includes variable numbers of CD133+ and EpCAM+ cells. We confirmed the propagation of CD44+ cells in samples from cases of human relapse, following standard PDAC treatment. Finally, using systemic administration of anti-CD44 antibodies in vivo, we demonstrated that CD44 is an efficient therapeutic target for treating tumor relapse, but not primary PDAC tumors. We conclude that CD44+ cells generate the relapsing tumor and, as such, are themselves promising therapeutic targets for treating patients with recurrent PDAC.